Project Summary/Abstract: Available treatments for diabetes mellitus are insufficient. Insulin gene therapy has the potential to improve long-term glycemic control in patients with diabetes. In rodents, delivery of an adenovirus containing an insulin transgene controlled by a metabolically responsive, liver specific promoter produces near normoglycemia. Blood sugars in hepatic insulin gene therapy (HIGT) treated rats normalize within two hours of an intra- peritoneal glucose load, and HIGT treated rats sustain prolonged periods of chow deprivation (24hrs) without lethal hypoglycemia. However, rodent physiology may not predict responses in humans, and the capacity of HIGT to impact diabetes associated hyperglycemia in non-rodent animals remains an open question. Swine are a more accurate model of human responses to diabetes mellitus than rodents. Like people, swine are omnivores and develop age related atherosclerosis that is accelerated by diabetes. We propose to demonstrate HIGT efficacy in pigs, and examine HIGT effects on whole body carbohydrate metabolism by nuclear magentic resonance spectroscopy (NMRS). In Aim 1 human derived promoter sequences will be created, and tested for metabolic responsiveness in rat, pig, primate, and human hepatocytes. This transgene optimization will provide preliminary data for extension of studies to primates. In Aim 2 we will establish hepatic gene transfer techniques in swine by depleting tissue macrophages and administering festral dilating agents prior to adenovirus. Invasive vascular monitoring, in addition to assessment of cytokine and lipogenic vascular mediators will guide additional interventions to improve tolerability. Gene marking studies will determine requisite doses of adenovirus to obtain ~25% hepatocyte transduction. In Aim 3 adenovirus will be used to deliver a metabolically responsive insulin transgene to diabetic pigs, and metabolic responsiveness following HIGT will be tested using intavenous glucose tolerance testing and fasting studies. In Aim 4 the effect of HIGT on hepatic glucose fluxes will be evaluated by serial 13-C NMRS studies, providing novel assessment of whole body carbohydrate metabolism in pigs, and quantifiying the utility of HIGT in pigs. Successful completion of these studies will provide an important model system for human diabetes, and may advance hepatic insulin gene therapy toward human studies. PUBLIC HEALTH RELEVANCE: Project Narrative: RELEVANCE TO VETERANS HEALTH The disease burden of diabetes mellitus among the veteran population is tremendous, costing the VA health system $1.5 billion each year 118. Approximately 20% of veterans have diabetes, and 70% are overweight, predisposing them to developing type 2 diabetes. The majority of veterans with diabetes will succumb to cardio- or cerebral vascular disease. HIGT inhibits endothelial dysfunction, an early marker of atherosclerotic vascular disease, in rats. However, whether HIGT can control glycemia and induce similar vascular effects in large mammals is disputed. This proposal utilizes STZ-diabetic pigs, a disease model with similarities to both type 1 and type 2 DM. Moreover, HIGT is effective in lowering BG in hyperglycemic Zucker rats (unpublished data), an accepted model of type 2 DM. Completion of this proposal will determine the efficacy of HIGT in pigs, and propel HIGT toward human trials, and potentially the treatment of tens of thousands of veterans.